Bone homeostasis and the skeletal structure are maintained by organized activity between osteoclasts for bone resorption and osteoblasts for bone formation. Osteoclasts, which are multinucleated cells, are differentiated from hematopoietic stem cells [T. Miyamoto, O. Ohneda, F. Arai, et al., Blood 98 (2001) 2544-2554]. The differentiation into osteoclasts is regulated by RANK ligand that is secreted from osteoblasts and activated T lymphocytes [Y. Y. Kong, U. Feige, I. Sarosi, et al., Nature 402 (1999) 304-309].
The receptor activator of nuclear factor kappa-B ligand (RANKL) binds to RANK, which is a receptor that is present in the osteoclast precursor, and osteoclast differentiation is induced in the presence of macrophage-colony stimulating factor (M-CSF). RANKL activates signaling pathways that regulate the formation of osteoclasts and the absorption of bones [J. Li, I. Sarosi, X. Q. Yan, et al., Proc. Natl. Acad. Sci. USA 97 (2000) 1566-1571]. RANK has no tyrosine kinase activity, and induces signaling through adaptor proteins, known as TNF receptor-associated factors (TRAFs) [L. Galibert, M. E. Tometsko, D. M. Anderson, et al., J. Biol. Chem. 273 (1998) 34120-34127]. TRAF6, which is a part of the intercellular molecule, plays a key role in the generation of osteoclasts and activates various downstream signals [M. A. Lomaga, W. C. Yeh, I. Sarosi, et al., Genes Dev. 13 (1999) 1015-1024]. TRAF6 binds to cytoplasmic domains of RANK, and then activates NF-κB and activator protein-1 (AP-1) [S. L. Teitelbaum, J. Clin. Invest. 114 (2004) 463-465].
Tumor necrosis factor-α (TNF-α), which is TNF ligand-based protein, is secreted in several types of cells including monocytes/macrophages or osteoclasts, and induces a number of biological responses through two cell-surface receptors termed TNFR1 and TNFR2 (also called TNFR p55 and TNFR p75, respectively). Both TNFR1 and TNFR2 induce intracellular signals that can stimulate the proteolytic breakdown of kappa B (IκB), a cytoplasmic inhibitor of NF-κB [Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van Antwerp, D., and Miyamoto, S. (1995) Genes Dev. 9, 2723-2735].
TNF-α modulates a varied range of responses, such as inflammation, immune modulation, cell proliferation and differentiation, and apoptosis [Ledgerwood, E. C., Pober, J. S., and Bradley, J. R. (1999) Lab. Invest. 79, 1041-1050]. TNF-α also promotes bone resorption in vitro and in vivo [Bertolini, D. R., Nedwin, G. E., Bringman, T. S., Smith, D. D., and Mundy, G. R. (1986) Nature 319, 516-518], and can induce the secretion of RANKL in osteoblasts [Hofbauer, L. C., Lacey, D. L., Dunstan, C. R., Spelsberg, T. C., Riggs, B. L., and Khosla, S. (1999) Bone 25, 255-259]. In addition, TNF-α is crucial to the pathogenesis of the bone and joint destructions that occur in rheumatoid arthritis, and has been implicated in the bone loss inperiodontitis, orthopedic implant loosening, and other forms of chronic inflammatory osteolysis. TNF-α is mediated by lipopolysaccharide-stimulated osteoclasts [Abu-Amer, Y., Ross, F. P., Edwards, J., and Teitelbaum, S. L. (1997) J. Clin. Invest. 100, 1557-1565]. TNF-α plays an important role in estrogen deficiency-induced bone loss in postmenopausal osteoporosis [Cenci, S., Weitzmann, M. N., Roggia, C., Namba, N., Novack, D., Woodring, J., and Pacifici, R. (2000) J. Clin. Invest. 106, 1229-1237].
Interleukin-3 (IL-3), which is the cytokine secreted mainly by activated T lymphocytes, may be used as a connection link between the immune system and the hematopoietic stem cell system [J. W. Schrader, Interleukin-3, in: A. W. Thomson, M. T. Lotze (Eds.), Academic Press, London, U K, 2003, pp. 201-225]. IL-3 directly acts on mouse osteoclast precursors, and promotes cell differentiation into macrophages, thereby inhibiting the RANKL-induced osteoclast differentiation [S. M. Khapli, L. S. Mangashetti, S. D. Yogesha, M. R. Wani, J. Immunol. 171 (2003) 142-151]. In the osteoclast precursor, IL-3 inhibits the phosphorylation and degradation of IκB, thereby preventing the nuclear translocation of NF-κB, which is induced by RANKL. In addition, IL-3 inhibits RANKL-induced c-Jun N-terminal kinase (JNK) activity, and down-regulates the expression of transcriptional factors, c-Fos and NFATc1. IL-3 inhibits the expression of RANK at the post-transcriptional stage, and this procedure was confirmed to be irreversible, through the in vivo experiment using mice.
Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosure of the cited papers and patent documents are entirely incorporated by reference into the present specification, and the level of the technical field within which the present invention falls, and the details of the present invention are explained more clearly.